Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. We are developing a mineralized collagen scaffold that mimics extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance pro-regenerative potential via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that each have displayed anti-inflammatory, immunomodulatory, and osteogenic properties. While potentially a powerful modification to our mineralized collagen scaffolds, the route of inclusion (matrix-immobilized or soluble) is not well understood. Here we compare the effect of introducing amnion and chorion membrane matrix versus soluble extracts derived from these membranes into the collagen scaffolds on scaffold biophysical features and resultant hMSC osteogenic activity. While inclusion of amnion and chorion matrix into the scaffold microarchitecture during fabrication does not influence their porosity, it does influence compression properties. Incorporating soluble extracts from the amnion membrane into the scaffold post-fabrication induces the highest levels of hMSC metabolic activity and equivalent mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG) compared to the conventional mineralized collagen scaffolds. Mineralized collagen-amnion composite scaffolds elicited enhanced early stage osteogenic gene expression (BGLAP, BMP2), increased immunomodulatory gene expression (CCL2, HGF, and MCSF) and increased angiogenic gene expression (ANGPT1, VEGFA) in hMSCs. Mineralized collagen-chorion composite scaffolds promoted immunomodulatory gene expression in hMSCs (CCL2, HGF, and IL6) while unaffecting osteogenic gene expression. Together, these findings suggest that mineralized collagen scaffolds modified using matrix derived from amnion and chorion membranes represent a promising environment conducive to craniomaxillofacial bone repair.

Craniomaxillofacial (CMF) bone injuries represent particularly challenging environments for regenerative healing due to their large sizes, irregular and unique defect shapes, angiogenic requirements, and mechanical stabilization needs. These defects also exhibit a heightened inflammatory environment that can complicate the healing process. This study investigates the influence of the initial inflammatory stance of human mesenchymal stem cells (hMSCs) on key osteogenic, angiogenic, and immuno­modulatory criteria when cultured in a class of mineralized collagen scaffolds under development for CMF bone repair. We previously showed that changes in scaffold pore anisotropy and glycosaminoglycan content can significantly alter the regenerative activity of both MSCs and macrophages. While MSCs are known to adopt an immuno­modulatory phenotype in response to inflammatory stimuli, here, we define the nature and persistence of MSC osteogenic, angiogenic, and immuno­modulatory phenotypes in a 3D mineralized collagen environment, and further, whether changes to scaffold architecture and organic composition can blunt or accentuate this response as a function of inflammatory licensing. Notably, we found that a one-time licensing treatment of MSCs induced higher immuno­modulatory potential compared to basal MSCs as observed by sustained immuno­modulatory gene expression throughout the first 7 days as well as an increase in immuno­modulatory cytokine (PGE2 and IL-6) expression throughout a 21-day culture period. Further, heparin scaffolds facilitated higher osteogenic cytokine secretion but lower immuno­modulatory cytokine secretion compared to chondroitin-6-sulfate scaffolds. Anisotropic scaffolds facilitated higher secretion of both osteogenic protein OPG and immuno­modulatory cytokines (PGE2 and IL-6) compared to isotropic scaffolds. These results highlight the importance of scaffold properties on the sustained kinetics of cell response to an inflammatory stimulus. The development of a biomaterial scaffold capable of interfacing with hMSCs to facilitate both immuno­modulatory and osteogenic responses is an essential next step to determining the quality and kinetics of craniofacial bone repair.

Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. Approximately 26% of injured Iraq war veterans sustained CMF injuries in the form of blast wounds, and 0.1% of births involve CMF defects like cleft palate. We previously developed a class of mineralized collagen scaffolds designed to mimic native extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance cellular bioactivity and osteogenic activity via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that individually have displayed anti-inflammatory, immunogenic, and osteogenic properties. They represent a potentially powerful compositional modification to the mineralized collagen scaffolds to improve bioactivity. Here we examine introduction of the placental-derived amnion and chorion membranes or soluble extracts derived from these membranes into the collagen scaffolds, comparing the potential for these modifications to improve hMSC osteogenic activity. We report structural analysis of the scaffolds via mechanical compression testing, imaging via scanning electron microscopy (SEM), and assessments of various metrics for osteogenesis including gene expression (Nanostring), protein elution (ELISA), alkaline phosphatase (ALP) activity, inductively coupled plasma mass spectrometry (ICP) for mineralization, and cell viability (AlamarBlue). Notably, a post fabrication step to incorporate soluble extracts from the amnion membrane induces the highest levels of metabolic activity and performs similarly to the conventional mineralized collagen scaffolds in regard to mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG). Together, these findings suggest that mineralized collagen scaffolds modified using elements derived from amnion and chorion membranes, particularly their soluble extracts, represent a promising environment conducive to craniomaxillofacial bone repair.

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